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PubMed2002

Characterization of aspartate aminotransferase isoenzymes from leaves of Lupinus albus L. cv Estoril.

Two aspartate aminotransferase (EC 2.6.1.1) isoenzymes (AAT-1 and AAT-2) from Lupinus albus L. cv Estoril were separated, purified, and characterized. The molecular weight, pI value, optimum pH, optimum temperature, and thermodynamic parameters for thermal inactivation of both isoenzymes were obtained. Studies of the kinetic mechanism, and the kinetics of product inhibition and high substrate concentration inhibition, were performed. The effect of some divalent ions and irreversible inhibitors on both AAT isoenzymes was also studied. Native PAGE showed a higher molecular weight for AAT-2 compared with AAT-1. AAT-1 appears to be more anionic than AAT- 2, which was suggested by the anion exchange chromatography. SDS-PAGE showed a similar sub-unit molecular weight for both isoenzymes. The optimum pH (between 8.0 and 9.0) and temperature (60-65 degrees C) were similar for both isoenzymes. In the temperature range of 45-65 degrees C, AAT-2 has higher thermostability than AAT-1. Both isoenzymes showed a high affinity for keto-acid substrates, as well as a higher affinity to aspartate than glutamate. Manganese ions induced an increase in both AAT isoenzymes activities, but no cooperative effect was detected. Among the inhibitors tested, hydroxylamine affected both isoenzymes activity by an irreversible inhibition mechanism.

Aspartate Aminotransferases↗
PubMed1976

[Distribution of creatine phosphokinase isoenzymes (EC 2.7.3.2) in cardiac cells].

The distribution of the creatinephosphokinase isoenzymes in the cardiac cells was studied by way of fractional extraction combined with an electrophoretic analysis of each of the received fractions. At the same time, the content of the creatinephosphokinase isoenzymes was studied in carefully purified preparations of myofibrils and mitochondria. The results of the conducted analysis indicate a heterogenic distribution of the creatinephosphokinase isoenzymes within the cells: about 30% of the cellular activity of the enzyme is contained in the mitochondria (the mitochondrial isoenzyme) 50% is comprised by the fraction dissolved in the cytoplasm (isoenzymes MM, MB and BB), and about 20%--in the myofibrils (isoenzyme MM), 5% of them being extracted only by a 0.9 M KCl solution. The myofibril preparation freed of the mitochondria also contains some important creatinephosphokinase activity (isoenzyme MM) comparable with their ATP-ase activity. The mitochondrial isoenzymes is found only in these structures of the cell.

Animals↗
PubMed2001

Lipopeptide adjuvants: generation of lactate dehydrogenase isoenzyme-specific antibodies for immunochemical diagnosis.

Lactate dehydrogenase catalyzes the final step in glycolysis, the interconversion of pyruvate and lactate. The tetrameric enzyme is composed of one or two subunits (H and/or M) resulting in five isoenzyme forms: LDH-H4, -H3M1, -H2M2, -H1M3, and -M4. The relative distribution of the LDH isoenzymes is tissue dependent and a significant marker for the diagnosis of hepatoma of the liver, myocardial infarction, muscular dystrophy, and a wide variety of other acute and chronic diseases to be detected by alterations of the LDH isoenzyme pattern in serum. Immunochemical approaches to the routine determination of LDH depend on isoenzyme specific antibodies. Since the H- and M-subunits for human LDH are highly homologous, LDH isoenzyme specific antibodies for immunochemical monitoring are hard to generate. Here we present data on the generation and characterization of LDH isoenzyme-specific mono- and polyclonal antibodies in different species in the presence of lipopeptide adjuvants. Western-Blot and ELISA analysis showed that antisera and monoclonal antibodies recognize their homologous antigens with high specificity and are therefore suitable for immunochemical monitoring of the LDH isoenzymes H4 and M4. In addition, they can be used for the determination of LDH isoenzyme specific activity which is an essential prerequisite for online amperometric immunosensor monitoring.

Adjuvants, Immunologic↗
PubMed1975

New instrument for rapid determination of activities of lactate dehydrogenase isoenzymes.

Lactate dehydrogenase isoenzymes can be distinguished kinetically by the fact that isoenzyme H is strongly inhibited a few seconds after the reaction is started if high concentrations of pyruvate are present, in contrast to the M isoenzyme. A new instrument that exploits this fact can measure both the total activity and the proportion of H isoenzyme in serum or plasma in 8 to 10 s. The instrument consists of a simplified stopped-flow apparatus in which the plasma is assayed for lactate dehydrogenase activity, and an electronic device that measures the rate of the reaction at two pre-set time intervals. The first rate is taken between 0.2 and 0.4 s after the reaction is started, a time at which both isoenzymes are fully active, and at which the rate obtained thus reflects total lactate dehydrogenase activity in the plasma sample. The second rate is measured 4 to 6 s after the start of the reaction, at which time the H isoenzyme has become inhibited and the observed rate compared to the initial rate is therefore proportional to the percentage of H isoenzyme activity in the serum. These two rates are electronically displayed on two three-digit voltmeters, the first display being the total activity, the second a number proportional to the inhibited slope. The percentage of M isoenzyme can then be calculated from the initial and final rate. A total of five to six repeat assays may be done within a minute on 1 ml of plasma or serum. This instrument may be of significant value in following the progress of myocardial infarctions and other diseases.

Autoanalysis↗
PubMed1992

Total leukocyte acid phosphatase and its isoenzymes in patients with leukemia.

Leukocyte acid phosphatase and its isoenzyme composition was studied in leukemic patients to determine the specificity of different isoenzymes in leukemic leukocytes. It was found that leukocyte acid phosphatase content is significantly increased in ALL, AML, and CML patients, while CLL patients had decreased levels of acid phosphatase. The distribution and intensity of leukocyte ACP isoenzymes vary in respective leukemic condition. Thus isoenzyme 'O' was predominant in AML and CML, while isoenzymes 1, 2 and 3 predominated in ALL. The lack of predominance of isoenzyme 3 was a feature in CLL patients. It was concluded that the isoenzyme patterns, though promising, presented inconclusive picture for diagnosis purpose and further studies on immunochemical characteristics of these isoenzymes are warranted to ascertain their cell specificity.

Acid Phosphatase↗
PubMed1992

Alkaline phosphatase isoenzymes in feline serum using an agarose gel alkaline phosphatase kit method.

Total serum alkaline phosphatase (ALP) activity is the product of the combined activity of isoenzymes from a number of tissue sources. In this study, a commercially available kit for electrophoretic separation of ALP isoenzymes in an agarose gel was used to separate ALP isoenzymes in feline tissue extracts and serum. Five separate bands of ALP activity were identified. These bands were numbered 1 to 5 with band 1 having the most anodal migration. The tissue of origin corresponding to the migration position of the isoenzymes are as follows: Band 3 was the liver isoenzyme, band 4 was the bone isoenzyme and ALP isoenzymes of both intestine and kidney migrate in the position labelled band 5. Band 1 appears to be related to albumin and does not represent true ALP activity. The tissue source of band 2 (a and b) was not identified. Serum ALP activity of mature, healthy cats is primarily of liver origin. Immature cats (< 1 year of age) have a greater proportion of the bone isoenzyme in the serum.

Alkaline Phosphatase↗
PubMed1992

Interconversion of E and S isoenzymes of horse liver alcohol dehydrogenase. Several residues contribute indirectly to catalysis.

The E and S isoenzymes of horse liver alcohol dehydrogenase differ by 10 amino acid residues, but only the S isoenzyme is active on 3 beta-hydroxysteroids. This functional difference was correlated to the differences in structures of the isoenzymes by characterizing a series of chimeric enzymes, which could represent intermediates in the evolution of catalytic activity. Deletion of Asp-115 from the E isoenzyme created the E/D115 delta enzyme that is active on steroids. The deletion alters the substrate binding pocket by moving Leu-116, which sterically hinders binding of steroids in the E isoenzyme. A chimeric enzyme (ESE) that has four changes in or near the substrate binding pocket (T94I/R101S/F110L/D115 delta) was 15-30-fold more catalytically efficient (V/Km) on uncharged steroids than was the E/D115 delta enzyme. Molecular modeling suggests that the substitutions at residues 94 and 110 indirectly affect the activity on steroids. ESE enzyme was 6-fold more active than the S isoenzyme on neutral steroids, due to substitutions not in the substrate binding pocket. The K366E and the Q17E/A43T/A59T substitutions in the S isoenzyme gave 2-fold increases in V/Km on steroids, which together can account for the changes observed with the ESE enzyme. The enzymes that are active on steroids did not bind 2,2,2-trifluoroethanol as tightly and were catalytically less efficient than the E isoenzyme with small alcohols. However, these enzymes were two to three and four to five orders of magnitude more efficient with 1-hexanol and 5 beta-androstane-3 beta,17 beta-diol, respectively, than with ethanol. These results demonstrate that several residues not directly participating in substrate binding or chemical catalysis contribute to catalytic efficiency.

Alcohol Dehydrogenase↗
PubMed2004

[Study of stability of lactate dehydrogenase isoenzymes in leucocytes of elderly people].

In this paper was investigated stability of isoenzymes of lactatdehydrogenase (LDH) of leukocytes of peripheral blood of persons at the age of 20 years (n=10) and more than 65 years (n=8). Extraction of the leukocytes was conducted by centrifugation of human blood on a double gradient of density. Leukocytes were destroyed by hypotonic shock in combination with vibration. Separation of LDH isoenzymes from destroyed leukocytes was conducted by the method of native electrophores. The revealing of activity of LDH isoenzymes was conducted by the method of restoration of nitroblue tetrasolium. Isoenzymes stability was evaluated according to alteration of activity after the incubation of destroyed leukocytes with urea in various concentrations (1M, 2M and 3M). Was shown the presence of three isoenzymes of LDG (LDH-1, LDH-2, LDH-3) in human leukocytes. As the age-dependent alteration of activity of isoenzymes of lactatdehydrogenase in human leukocytes was shown. It can be caused by age-related changes of physico-chemical properties of leukocytes' membranes and/or isoenzymes stability. Stability of LDH-3 was decreased with the age. Observed decrease of LDH-3 stability can be caused by age-related modification of M-subunits of the studied isoenzymes.

Adult↗
PubMed1991

Effects of azamethiphos, an organophosphorus insecticide, on serum cholinesterase activity and isoenzymes in the rat.

The inhibitory effect of azamethiphos (Az), an organophosphorus insecticide, was detected in both the serum cholinesterase (ChE) activity and the ChE isoenzyme concentration in male Wistar rats. In our highest Az dosage group (270 mg/kg), following a single po administration, the serum ChE activity was significantly inhibited at 6 and 24 h post-Az administration. Five days after Az treatment, the ChE activity was still inhibited, but the observed difference was not statistically significant. The ChE activity increased to levels comparable to the control group by day 10. For the other Az-dosed groups, the ChE activities were inhibited only 6 h following administration. The serum ChE isoenzymes for all Az-dosed and control groups had 6 bands, indicating that Az did not effect the number of isoenzyme bands, indicating that Az did not effect the number of isoenzyme bands. However, Az had a significant effect on the ratios of the concentrations of these isoenzyme bands. The ratio of the main ChE isoenzyme bands (bands 5 and 6) was band 6 greater than band 5 for the young control group. As our control group aged, this ratio was reversed, with band 5 greater than band 6. For the highest Az dosage group (270 mg/kg), the ChE isoenzyme band ratio was band 5 greater than band 6 6 h after Az dosing. This proportion of band 6 decreased in parallel with an increase in band 5 during our 30-d experimental period. A correlation between the total ChE activity and ChE isoenzyme band 6 was found.

Animals↗
PubMed1990

Creatine kinase and lactate dehydrogenase isoenzyme patterns in cultured normal and pathological gingival fibroblasts.

The isoenzyme patterns of creatine kinase (CK) and lactate dehydrogenase (LDH) were examined in cultured fibroblasts derived from the gingival tissue of a healthy person and patients with periodontitis and gingival fibromatosis. Human foreskin fibroblasts were also included for comparison. Cytosol fractions were prepared by sonication and centrifugation. Gingival fibroblasts from normal subjects (A), and from patients with periodontitis (B) or fibromatosis (C), exhibited CK-MM (muscle form) as a predominant isoenzyme and CK-BB (brain form) as a minor fraction. There was no significant difference in specific activities of CK-BB fraction in all 3 types of fibroblasts. However, B fibroblasts contained a higher proportion of MM type isoenzyme than A fibroblasts. C fibroblasts showed the lowest amount of CK-MM isoenzyme as compared to either A or B fibroblasts. In contrast, fibroblasts derived from human foreskin (D) contained only a small amount of CK-BB isoenzyme, but CK-MM was not present. In the studies of LDH isoenzymes, A, B, and C fibroblasts possessed only 3 isoenzymes, namely, LDH-3, LDH-4 and LDH-5. In terms of the relative abundance of M vs H subunits, A, B, and C fibroblasts were shown to have a preponderance of M subunits (85-87% M vs 13-15% H subunits). In comparison, D fibroblasts contained 4 isoenzymes (LDH-2, LDH-3, LDH4, LDH-5) with 73% M and 27% H subunits. The ratio of LDH-5 to LDH-4 was calculated for all fibroblasts tested, and the values were 1.11 in A, 1.39 in B, 1.16 in C and 0.64 in D.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1988

Substrate specificity of rat liver glutathione S-transferase isoenzymes for a series of glutathione analogues, modified at the gamma-glutamyl moiety.

The substrate specificity of purified rat liver glutathione S-transferases (GSTs) for a series of gamma-glutamyl-modified GSH analogues was investigated. GST isoenzyme 3-3 catalysed the conjugation of 1-chloro-2,4-dinitrobenzene with six out of the nine analogues. alpha-L-Glu-L-Cys-Gly and alpha-D-Glu-L-Cys-Gly showed catalytic efficiencies of 40% and 130% that of GSH respectively. The GSH analogue with an alpha-D-glutamyl moiety appeared to be a highly isoenzyme-3-3-specific co-substrate: kcat./Km with GST isoenzyme 4-4 was only about 5% that with GST isoenzyme 3-3, and no enzymic activity was detectable with GST isoenzymes 1-1 and 2-2. GST isoenzyme 4-4 showed some resemblance to GST 3-3: five out of nine co-substrate analogues were accepted by this second isoenzyme of the Mu multigene family. Isoenzymes 1-1 and 2-2, of the Alpha multigene family, accepted only two alternative co-substrates, which indicates that their GSH-binding site is much more specific.

Animals↗
PubMed1988

Diagnostic value of alkaline phosphatase isoenzyme separation by affinity electrophoresis in the dog.

Affinity electrophoresis, using wheat germ lectin, was used to separate the alkaline phosphatase isoenzymes in the sera of 150 dogs with alkaline phosphatase values greater than or equal to 150 IU/L. The method provided clearer separation of the liver, bone and steroid-induced alkaline phosphatase isoenzymes commonly observed in canine serum, compared to conventional cellulose acetate electrophoresis. The dogs were divided into four patient groups determined by previous corticosteroid treatment, evidence of elevated endogenous corticosteroid levels, age and alanine aminotransferase values. The isoenzyme pattern of each patient was qualitatively assessed. The isoenzyme pattern most frequently observed was greater than 50% steroid induced alkaline phosphatase, which was present in 76 of 150 dogs. This pattern was observed in 18 of 22 dogs receiving corticosteroid therapy, two of three dogs with hyperadrenocorticism, and in dogs with a variety of other diagnoses. The majority of immature dogs (12 of 20) had an isoenzyme pattern consisting of greater than 50% bone. The majority of dogs with active hepatocellular injury (16 of 27) had greater than 50% liver isoenzyme. The isoenzyme pattern was not specific for certain diseases, therefore the diagnostic usefulness is limited. However the isoenzyme result is useful in some cases to determine which further diagnostic tests are indicated, and to determine the source of alkaline phosphatase elevation.

Adrenal Cortex Hormones↗
PubMed1984

[Shift of placental alkaline phosphatase isoenzymes in the course of pregnancy].

It is generally accepted that every placental protein shows its characteristics vicissitude pattern during pregnancy and that alkaline phosphatases (ALP) are subdivided into three kinds of isoenzymes, i.e.: tissue-unspecific, intestinal and termplacental ALPs. In this study, the changes in ALP isoenzyme profile throughout the course of pregnancy was studied by biochemistry, enzyme- and immuno-histochemistry, and the following results were obtained. In early pregnancy, ALP activity was composed mostly of tissue-unspecific ALP isoenzyme, and its activity revealed a peak value around 10 weeks of pregnancy as in the case of the hCG serum concentration. At the end of the second trimester of pregnancy, instead of tissue-unspecific isoenzyme, ALP activity was mainly composed of term placental ALP isoenzyme, but a very small amount of the isoenzyme was proved to exist in the villi at the 6th week of gestation. The above results were confirmed by morphological studies such as enzyme-cytochemistry and immuno-cytochemistry. It was therefore concluded that at least two genes of ALP isoenzymes existed in the villi and that the regulation of gene activity changed during the course of pregnancy. Furthermore, since tissue-unspecific ALP isoenzyme showed a pattern of change similar to that of serum hCG, the existence of a developmental phase-specific gene set was postulated.

Alkaline Phosphatase↗
PubMed1984

Myosin isoenzyme expression in rat ventricle: effects of thyroid hormone analogs, catecholamines, glucocorticoids and high carbohydrate diet.

The effects on ventricular myosin isoenzyme expression of naturally occurring and synthetic thyroid analogs (3,5,3'-L-triiodothyronine, 3,5,3'-D-triiodothyronine, 3,3',5'-L- triiodothyronine , 3,5,3'-L-triiodothyroacetic acid and 3,5-L-diiodothyronine), catecholamines and high carbohydrate diets have been studied in thyroidectomized and hypophysectomized rats. Also, the effects on myosin isoenzyme expression of adrenalectomy and hydrocortisone replacement have been studied in euthyroid animals. Myocardial CO2 production and hepatic alpha-glycerolphosphate dehydrogenase activity were measured to monitor the effects of these interventions on tissue respiration. The results indicate that there was no significant separation between the actions of thyroid analogs on metabolic parameters and myosin isoenzyme patterns. However, high carbohydrate feeding in hypophysectomized rats increased the isoenzyme V1 from 12% to about 36% of total myosin; partial replacement with 3,5,3'-L-triiodothyronine and fructose feeding had synergistic actions. In thyroidectomized rats, feeding a high carbohydrate diet increased the V1 form from undetectable levels to about 28% of total myosin; partial 3,5,3'-L-triiodothyronine replacement had an additive effect. Beta adrenergic stimulation with isoproterenol and blockade with propranolol did not affect myosin isoenzyme expression. Adrenalectomy in euthyroid rats caused a 33% decrease in the V1 form and a corresponding increase in the V3 isoenzyme, which could be reversed by treatment with hydrocortisone. Thus, thyroid analogs do not selectively stimulate myosin isoenzyme expression as compared with their effects on energy production. Furthermore, the results suggest that the mechanism for regulation of cardiac myosin isoenzymes may involve a primary signal related to dietary carbohydrate, which is modulated by thyroid hormone, and possibly glucocorticoids.

Animals↗
PubMed1983

Lactate dehydrogenase and creatine kinase isoenzyme levels in the tissues and serum of normal lambs.

The normal activities of lactate dehydrogenase and creatine kinase isoenzymes in the tissues and serum of clinically normal five-and-a-half-month-old lambs are presented. The percentage distribution of the lactate dehydrogenase isoenzymes in serum, liver, heart, lung and skeletal muscle were in agreement with previous studies but the distribution in ovine abomasum, small intestine, large intestine and red blood cells has not been previously described. Up to five creatine kinase isoenzymes were detected in ovine tissues and four in serum. One creatine kinase isoenzyme present in serum and tissues was thought to represent a mitochondrial isoenzyme which is absent from the normal serum of other species. Estimations of both lactate dehydrogenase and creatine kinase isoenzymes allowed all the tissues examined to be distinguished and were therefore more likely to allow tissue-specific isoenzyme patterns to be detected in serum than the estimation of the isoenzymes of either enzyme alone.

Animals↗
PubMed1984

Atypical alkaline phosphatase isoenzyme in serum from a patient with Hodgkin's disease.

An alkaline phosphatase isoenzyme that did not move from the origin in agarose gel electrophoresis was detected in serum from a 51-year-old woman with Hodgkin's disease. Inhibitor and heat-inactivation studies of the patient's serum alkaline phosphatase showed properties resembling those of both liver and bone isoenzymes. No immunoglobulin or high-molecular-mass complexes with the alkaline phosphatase isoenzyme were detected. The relative molecular mass (Mr) of the atypical alkaline phosphatase isoenzyme was 182 000, that of the liver alkaline phosphatase isoenzyme control 170 000. Treatment of both of these isoenzymes with neuraminidase gave a product with an Mr of 140 000. We propose that a post-translational modification increased the carbohydrate content of the liver alkaline phosphatase isoenzyme, thus changing the charge characteristics of the enzyme and decreasing its electrophoretic mobility. We believe this to be the first report of a post-translational modification in a heat-sensitive isoenzyme of alkaline phosphatase.

Alkaline Phosphatase↗
PubMed1981

Serum levels of creatine phosphokinase and its isoenzymes in normal and stressed neonates.

Twenty-six normal newborns (13 male, 13 female) with normal prenatal histories, no perinatal stress, and normal vaginal deliveries had creatine phosphokinase (CPK) activity and isoenzyme activities assayed in cord blood and in 24-hour postpartum serum. Total CPK activity was high in cord blood when compared with adult control values. Moreover, the total CPK was significantly higher in serum at 24 hours of age compared with cord blood. There was a significant increase in both the skeletal muscle isoenzyme and the cardiac muscle isoenzyme from birth to 24 hours of age. There was a decrease in the brain isoenzyme at 24 hours of age which was not statistically significant. These results were compared with values obtained in a group of 10 neonates with severe cardiac problems. Three of the ill neonates had significant elevation of total serum CPK and the skeletal muscle isoenzyme when compared with the normal newborns. There were no significant differences between the normal infants and the ill neonates for the cardiac isoenzyme and the brain isoenzyme. These data suggest that caution should be used in the diagnosis of certain neonatal cardiac syndromes based on serum CPK levels and isoenzyme alone.

Adult↗
PubMed1980

Creatine kinase radioimmunoassay and isoenzyme electrophoresis compared in the diagnosis of acute myocardial infarction.

We compared, in 116 patients, the relative usefulness of results of tests for creatine kinase B isoenzymes, as measured by radioimmunoassay, and the MB isoenzyme, as measured by electrophoresis, in diagnosis of acute myocardial infarction. The radioimmunoassay was specific for isoenzymes of creatine kinase containing the B subunit. All patients with acute transmural infarcts had positive test results by both techniques, but concentrations of B-isoenzymes were more frequently above normal than were MB bands in the case of patients with acute subendocardial infarcts and in the case of all patients with acute myocardial infarcts from whom sera were collected more than 24 h after onset of chest pain. Concentrations of B-isoenzymes also were increased, even when MB bands were not electrophoretically detectable, in specimens from several patients without documented actue myocardial infarcts. These abnormal results presumably were caused by increased concentrations of the BB isoenzyme in serum. Accordingly, an increased concentration of B-isoenzymes had less diagnostic specificity and predictive value for acute myocardial infarction than did a detectable MB band. Results of isoenzyme electrophoresis were more reliable for establishing this diagnosis, but the results of radioimmunoassay were more reliable for excluding it in patients with chest pain as the primary symptom.

Clinical Enzyme Tests↗